Adeno-Associated Viruses (AAVs) are the most commonly used gene therapy vector and AAV technology is
posed to deliver new treatments for a wide range of previously incurable conditions. AAVs are non-pathogenic
to humans and infect non-dividing cells, making them ideal vectors for targeted regulation of gene expression in
the human central and peripheral nervous system (CNS/PNS). These highly attractive factors have broadly
increased interest in therapeutic AAVs; however, existing AAV vectors require very high doses to express
transgenes in CNS/PNS targets at levels that mitigate pathology. The resulting dose-dependent toxicities in other
organs have led to clinical failures and unpredictable off-target delivery, which is a major limitation of modern
gene therapy. There is a critical need for high-throughput, comprehensive profiling of viral tropism and
transduction efficiency that could speed development of safe and effective AAVs.
AAV tropism is typically determined using immunohistochemical techniques, which are the gold standard for
detection of transgene expression. However, these methods are labor-intensive and permit testing of only a
small number of pre-determined tissues. Efficient and comprehensive AAV tropism profiling technologies would
facilitate discovery of AAV variants with greater CNS/PNS tropism by enabling fast iterative testing and direct
comparison of AAV mutant vectors. My long-term goal is to develop novel gene therapy treatments for peripheral
nerve injuries. The objective of this proposal is to test feasibility of using a custom hyperspectral whole-body
imaging cryomacrotome (BioSlice) for rapid, automated evaluation of AAV tropism for the nervous system.
Our proposed experiments combine two powerful technologies—transgenic fluorescent reporter mice and
whole-body, high-resolution hyperspectral wide-field imaging—to detect AAV transduction and provide a new
approach to AAV tropism profiling. In Aim 1, we will use the BioSlice to search for off-target delivery in Ai14 mice
that have been injected with three AAV variants that have known tropism for the CNS/PNS. In Aim 2, we will test
whether the BioSlice can detect multiplexed AAVs using distinct fluorescent reporters in the same animal, and
examine effects on AAV tropism related to injection site.
Completion of these aims will deliver an innovative application of an advanced imaging technology to address
a fundamental bottleneck in AAV development. If successful, our work will provide robust proof-of-concept that
the BioSlice can be used to rigorously evaluate pre-clinical AAVs for therapeutic use. This transdisciplinary
approach has the potential to enable tropism discovery through unbiased investigation of all tissues in the mouse,
thereby accurately modeling potential off-target delivery and negative side-effects across AAV serotypes,
expression cassettes and injection sites.